**1. Introduction**

Honey has been described as a natural sweet mixture produced by honeybees from the nectar of flowers or from living parts of plants. Bees combine this mixture with substances of their own, and then it is deposited, dehydrated, and stored in the honeycomb for further uses [1]. Honey is the most characterized bee product due to its nutritional value as a natural. Honey is composed of several carbohydrates, mainly fructose and glucose (85–95% of total sugars). Glucose has a lower degree of solubility than fructose. The ratio of glucose to fructose determines the liquid state of a given honey. Other types of sugar are present due to the union of two or more molecules of fructose or glucose as polysaccharides. Additionally, certain substances are available in honey, such as organic acids, amino acids, proteins, enzymes, lipids, flavonoids, and vitamins that are responsible for its biological properties including antioxidant or antibiotic activities [2].

Additionally, certain substances are available in honey, such as organic acids, amino acids, proteins, enzymes, lipids, flavonoids, and vitamins that are responsible for its biological properties including antioxidant or antibiotic activities [3, 4].

Melissopalynological analysis is used to establish whether a honey is unifloral or not. Unifloral honey has a higher market price because at least 45% of the pollen

grains in its solids are from the same plant species. Therefore, the quality of a honey depends on the presence and concentration level of specific compounds and the botanical origin classification [5].

Honey can obtain the characteristics of plants whose pollen grains and nectar have been taken by bees. Thus, the biological properties are related to the plant species and its attributes [6]. Antioxidant activity is one of the observed biological properties of honey. The presence of enzymatic antioxidants (glucose oxidase, catalase) and non-enzymatic antioxidants (flavonoids, ascorbic acid, and phenolic acids) have been detected in many honeys [7, 8]. Several studies have looked to establish some relationship between phenolic compounds and the antioxidant properties of honey. An analysis of the phenolic compounds profile of unifloral Rhododendron honey produced in Turkey demonstrated that increased antioxidant activity was related to higher concentrations of those molecules. The same effect was observed for the antibacterial capabilities of honey samples [9, 10].

The identification of phenolic compounds includes many extraction techniques that permit the isolation of the phenolic fraction from the rest of the honey's components. Solid-Phase Extraction (SPE) procedures are recommended for cleaning the samples, followed by High-Performance Liquid Chromatography (HPLC) or Capillary Electrophoresis, CE [11]. Those techniques have been used to determine the chemical profiles of natural products from extracts obtained from complex organic matrices such as honey. Despite its high resolving power, highperformance liquid chromatography (HPLC) may present some limitations for the separation of molecules belonging to the same family, even when proper sample cleaning is performed to achieve better results. In the same way, capillary electrophoresis and the related technique, electrokinetic chromatography (EKC), in zone format (CZE) allow for the analysis of ionic and neutral compounds on the same column. The great advantage of this methodology is the amount of sample needed for each analysis; it requires only a few nanoliters of extract with a solvent waste of 1 mL–2 mL per assay [12]. Several research studies have focused on the identification of phenolic compounds in bee products. Samples of commercial propolis were studied using CE, and 15 polyphenols were separated with a buffer of sodium tetraborate 30 mM, pH 9.0, and under an applied voltage of 15 kV. Borate buffers form complexes with orthodihydroxyl groups on the flavonoid skeleton and facilitate separation. In the same study, three different extracts were produced (ethanolic, aqueous-ethanolic, and aqueous-glycolic extracts) to compare the levels of available analytes in each one. After this procedure, it was possible to establish a reproducible fingerprint of the polyphenolic profiles, the pattern of which depended on the nature of the extraction solvent [13].

By the way, the determination of the antioxidant capability of honey requires UV– Vis determinations. For instance, the colorimetric assays for the general quantification of phenolics is done by Folin–Ciocalteu reaction; assessment of radical scavenging using the reduction reaction of the radical 2,2-diphenyl-1-picrylhydrazyl (DPPH) is a very helpful method for this aim [3]; the assessment of antioxidant activity using the ferric reducing/antioxidant power assay (FRAP) [14, 15] and the oxygen radical absorbance capacity (ORAC) [16] has been frequently used.

#### **2. Chilean honey**

In 2020, according to figures obtained from Agricultural and Livestock Service of Chile, there are 8777 beekeepers who manage 1.241,504 beehives distributed throughout the country [17]. In that sense, 1991 tons were exported, and most of the honey was sent to European markets, with Germany being the main buyer,

followed by Belgium. Also, China and the United Arab Emirates have also emerged as important buyers of Chilean honey. Chilean bee products have interesting biological properties that improve their natural potential as an attractive exportable nutritional food. Chemical characterization is necessary for certifying their natural attributes. Furthermore, chemical content analysis enables the fulfillment of international regulations for healthy and safe foods because those markets are very strict in terms of the food safety issues. Several studies on the potential properties of native unifloral honey have been conducted. The Ulmo Honey (Eucryphia cordifolia) demonstrated the greatest antibacterial power among the selected Chilean unifloral honey samples. The main identified compounds are gallic, caffeic, coumaric, and chlorogenic acids [18, 19].

## **3. Undesirable residues**

The presence of undesirable compounds in honey occurs when beehives or plants are exposed to pollutants caused by human activities; in this case, the final composition of honey is modified, and the effectiveness of its biological activity changes. Recently, it has been demonstrated that honey has a specific chemical profile of inorganic elements related to the place where it was produced [20]. This information enables the certification of the geographical origin of a honey [21]. Similarly, studies showed that the inorganic content is not dependent on the botanical origin, but rather on the composition of the soils and water in the areas surrounding beehives, and other environmental conditions play an important role in this case [10, 22]. Also, honeys that contained metals in their composition showed a decreased antioxidant activity compared with control samples [23]. The same trend was found in bee pollen samples obtained from the same beehives [24]. Furthermore, it was possible to observe that the chemical behavior of phenolic compounds was modified due to the presence of metals, based on analyses by capillary electrophoresis with diode array detector (CE-DAD) [12].

## **4. Good agricultural practices (GAP)**

According to the Food and Agricultural Organization of the United Nations (FAO), the current definition of Good Agricultural Practices (GAP) includes codes, standards, and regulations that have been developed in recent years by the food industry and producers' organizations but also governments and nongovernmental organizations, aiming to codify agricultural practices at farm level for a range of commodities. Their purpose varies from fulfillment of trade and government regulatory requirements (in particular with regard to food safety and quality), to more specific requirements of specialty or niche markets. The objective of these GAP codes, standards, and regulations includes, to a varying degree: ensuring safety and quality of produce in the food chain; capturing new market advantages by modifying supply chain governance; improving natural resources.

Currently, despite those findings, the standard methods for the measurement of those compounds still include an analysis by HPLC with mass spectrometry (MS). The efficiency of mass spectrometry and the optimization of chromatographic procedures have helped to decrease the experimental time for each analysis. Moreover, several antibiotics can be detected in just one chromatogram. This was the case in a study that was performed by selecting 11 honey samples from different botanical origins produced in Granada, Spain. After HPLC coupled to electrospray ionization (ESI) time-of-flight (TOF) mass analyzer, the following antibiotics were quantified with an average of MRL from 0.05 to 0.76 μg Kg<sup>1</sup> and a run time of approximately 11 minutes: chlortetracycline, demeclocycline, doxycycline, methacycline, minocycline, oxytetracycline, tetracycline, and rolitetracycline [25].
